In a method of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, which is known as GSD (Ground State Depletion) scanning fluorescence light microscopy, the luminophore, by means of GSD light comprising a local minimum, via its excited electronic luminescent state, like, for example, via its excited electronic singlet state, is transferred into a dark state, like, for example, into a long living triplet ground state, out of which it is not excited into the luminescent state by luminescence excitation light. Everywhere outside the local minimum of the intensity distribution of the luminescence inhibiting light, this transfer is saturated. I.e. only in the local minimum of the intensity distribution of the luminescence inhibiting light, the luminophore, after subjection to the luminescence inhibiting light, is still in its electronic ground state out of which it is excited into the luminescent state by means of the luminescence excitation light. Luminescence light emitted by the luminophore after excitation by the luminescence excitation light thus exclusively stems from the local minimum of the intensity distribution of the luminescence inhibiting light and may thus be assigned to the position of the local minimum within the sample.
In the method known as GSD, there is a considerable danger of bleaching the luminophore, because the luminophore, both in its long living dark state into which it is transferred by the luminescence inhibiting light, and in the excited electronic luminescent state in which it is temporarily during its transfer into its dark state, to an increased extent tends to chemical reactions, like, for example, with oxygen, and/or is prone to the danger that it is further electronically excited by the luminescence inhibiting light or the luminescence excitation light so that photochemical bleaching of the luminophore occurs.
A further method of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, which is known as a variant of RESOLFT (Reversible Saturable Optical Fluorescence Transitions) scanning fluorescence light microscopy, makes use of so-called switchable luminophores. These luminophores, by means of luminescence inhibiting light in the form of switching off light, are switchable out of a first conformation state, i.e. a first atomic configuration, in which they are active as luminophores into a second conformation state in which they are not active as luminophores, i.e. in which they are, at least by means of the luminescence excitation light which, in the first conformation state is suitable for exciting the luminescent state, not excitable into a luminescent state in which they emit the luminescence light registered as the measurement signal. With a sufficient long lifetime of the second conformation state, only comparatively low light intensity distributions are necessary to saturate such a switching process everywhere outside the local minimum of the intensity distribution of the luminescence inhibiting light. Further, there is no significant danger that the luminophore transferred into its other conformation state bleaches out of this other conformation state, as the luminophore does not respond to the luminescence inhibiting light and the luminescence excitation light in this conformation state. However, the transfer of the switchable luminophore into its second conformation state by means of the luminescence inhibiting light also takes place via an excited electronic state which, with high intensities of the luminescence inhibiting light, may be a starting point for photochemical bleaching of the luminophore. For this and other reasons, the absolute number of usable switching processes between the two conformations states are limited with a plurality of switchable luminophores, particularly if they are switched actively, i.e. by means of luminescence enabling light, out of their second not luminescence-able conformation state into their first luminescence-capable conformation state. Further, the already mentioned long lifetimes of the conformation states, even if one actively switches back and forth between the conformation states, mean that the methods of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, which are known as RESOLFT, are comparatively slow. Finally, the number of commercially available switchable luminophores which are suitable for marking structures of a sample is limited, particularly, when compared to the high number of generally available luminophores. The development of new stable switchable luminophores is also laborious.
In a method of spatial high resolution imaging of a structure of a sample, the sample comprising a luminophore, which is known as STED (Stimulated Emission Depletion) scanning fluorescence light microscopy, the sample, in a measurement area, is at first subjected to luminescence excitation light which excites the luminophore out of an electronic ground state into a luminescent state. Then, the sample, in the measurement area, is subjected to an intensity distribution of STED light comprising a local minimum, which de-excites the excited luminescent state by stimulated emission back into the ground state. If the luminescence inhibiting light has de-excited the luminescent state everywhere outside the minimum by stimulated emission, luminescence light emitted out of the measurement area afterwards may only stem out of the local minimum of the intensity distribution of the luminescence inhibiting light and may thus be assigned to the position of the local minimum within the sample.
In the method known as STED, a very high spatial resolution in imaging a structure of a sample, the structure comprising a luminophore, is actually achieved. Here, however, the luminophore is considerably stressed photochemically and thus tends to bleach. The reason is that the luminescence inhibiting light which has to be applied at a high absolute intensity for narrowing down the local minimum in the form of a zero point of its intensity distribution acts upon the luminophore already being in its excited luminescent state. Thus, besides the desired stimulated emission which transfers the luminophore back into its ground state, other processes, particularly farther reaching electronic excitations resulting into bleaching of the luminophore, are not unlikely. Even a new excitation of the luminophore at first de-excited by stimulated emission may occur due to the light originally provided for luminescence inhibition.
All known methods of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, are based on the fact that the luminescence inhibiting light transfers the luminophore either out of its luminescent-capable ground state via an excited electronic state into a dark state prior to application of the luminescence excitation light, or out of the excited luminescent state back into the ground state. Thus, in each case, the luminophore is subjected to luminescence inhibiting light of high intensity that has a wavelength in the absorption spectrum of the luminophore, and an excited electronic state which is associated with the danger of photochemical bleaching of the luminophore is involved in enhancing the spatial resolution by means of the luminescence inhibiting light. Further, there seems to be a correlation between the lifetime of the dark state and the necessary intensity of the luminescence inhibiting light which may be the lower the longer the lifetime of the dark state.
There still is a need of a method of spatial high resolution imaging of a structure of a sample, the structure comprising a luminophore, in which the luminophore is subjected to a particularly low danger of photochemical bleaching and which nevertheless allows for high velocities in scanning the sample with the minimum of the intensity distribution of the luminescence inhibiting light.